bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

TPX2 activation by GM130 controls astral formation and spindle orientation

Haijing Guo1, Jen-Hsuan Wei2 and Joachim Seemann1*

1 Department of Cell Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA

2 Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

* correspondence: [email protected]

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Abstract

Spindle orientation is important in multiple developmental processes as it determines cell fate

and function. The correct orientation of the mitotic spindle depends on the assembly of a proper

astral microtubule network. Here, we report that the spindle assembly factor TPX2 regulates the

astral microtubule network. TPX2 in the spindle pole area is activated by GM130 on mitotic

Golgi membranes to promote astral microtubule growth. GM130 relieves TPX2 inhibition by

competing for α binding. During mitosis, phosphorylation of importin α at Serine 62 by

Cdk1 switches its substrate preference from TPX2 to GM130, thereby enabling competition-

based activation. Importin α S62A impedes local TPX2 activation and compromises astral

microtubule formation, ultimately resulting in misoriented spindles. Blocking the GM130-

importin α-TPX2 pathway impairs astral microtubule growth. Our results reveal a novel role for

TPX2 in the organization of astral . Furthermore, we show that the substrate

preference of the important mitotic modulator importin α is regulated by Cdk1 phosphorylation.

Keywords

mitosis, aster microtubules, spindle orientation, TPX2, importin α, GM130, CDk1,

phosphorylation, Golgi

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Introduction

Cell proliferation requires accurate partitioning of all vital cellular constituents during cell

division. In eukaryotic cells, the microtubule-based mitotic spindle machinery delivers a precise

set of into each daughter cell. Spindle microtubule filaments are further

responsible for the correct positioning and orientation of the spindle to define the cell division

plane, which plays a critical role in cell fate decision, morphogenesis and maintenance of tissue

organization (Bergstralh et al., 2017). Consequently, aberrant spindle orientation causes defects

in various physiological processes such as gastrulation, neuronal differentiation, epithelial self-

renewal and tissue stratification (Gong et al., 2004; Lechler and Fuchs, 2005; Fish et al., 2006;

Cabernard and Doe, 2009). Determination and maintenance of spindle orientation largely rely on

the mitotic microtubule network and its connections to the cell cortex.

Assembly of the mitotic spindle is tightly controlled in space and time through regulated

and polymerization (Prosser and Pelletier, 2017). In higher eukaryotes,

mitotic microtubule arrays predominantly originate at the , which form the spindle

poles. Astral microtubules emanate from the spindle poles and extend to the cell cortex. This

spindle-cortex connection is required to generate the pulling forces that dictate positioning and

orientation of the spindle. Reductions of astral microtubules weaken connections between the

spindle and the cell cortex, which can often lead to spindle oscillation and misorientation (Petry,

2016). Though astral microtubules are important in spindle positioning, microtubule filaments

connected to kinetochores are directly engaged in the segregation of chromosomes into the

daughter cells. Kinetochore microtubules are bundles of parallel microtubule filaments known as

K-fibers that originate at the spindle poles and attach to the chromosomes via kinetochores.

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Proper attachment of all K-fibers in the bipolar spindle is required to silence the spindle

assembly checkpoint and allow progress into anaphase (Prosser and Pelletier, 2017).

In early mitosis, kinetochore microtubules are nucleated at the . This key step of

spindle formation ensures that all chromosomes are captured by the kinetochore microtubules,

with this process being regulated by the small GTPase . Upon loading with GTP (via its

chromatin-localized guanine nucleotide exchange factor RCC1), RanGTP destabilizes ternary

complexes of classical nuclear localization sequence (NLS)-containing plus importin

α/ß and promotes their dissociation (Carazo-Salas et al., 1999). Through this mechanism,

RanGTP relieves importin α-mediated inhibition of essential spindle assembly factors to promote

microtubule nucleation, stabilization and spindle organization (Kalab and Heald, 2008). One of

the key spindle assembly factors is the microtubule nucleator TPX2. Once released from

importin α inhibition, TPX2 activates and stabilizes active by shielding it from

dephosphorylation (Bayliss et al., 2003). The Aurora A-TPX2 complex binds to microtubules,

further enhancing microtubule polymerization at the chromosomes (Kufer et al., 2002; Anderson

et al., 2007).

In addition to functioning at chromatin, the microtubule nucleation activity of TPX2 can also

occur at mitotic Golgi membranes through the Golgi-resident membrane GM130 (Wei et

al., 2015). During mitosis, the single copy of the mammalian Golgi apparatus is disassembled

into a collection of vesicles that cluster and localize in the spindle pole area (Shima et al., 1998).

These mitotic Golgi clusters are subsequently delivered by the spindle into the daughter cells

where the membranes fuse to re-establish a contiguous Golgi structure (Wei and Seemann, 2017;

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Mascanzoni et al., 2019). Mitotic disassembly of the Golgi is initiated by Cdk1-mediated

phosphorylation of the Golgi-resident membrane protein GM130 (Lowe et al., 2000), which

exposes its NLS motif that sequesters importin α from TPX2 by direct competition. TPX2 is

thereby activated to locally drive microtubule nucleation at mitotic Golgi membranes. The

nascent microtubules are then captured by GM130, which couples Golgi membranes to the

spindle (Wei et al., 2015). However, it had remained unclear how TPX2 is preferentially

liberated from importin α by the NLS motif of GM130, given the high abundance of NLS-

containing proteins that are released upon nuclear envelope breakdown during mitotic entry.

Furthermore, how importin α regulated this event spatiotemporally so that the process could be

coordinated with other microtubule nucleation activities to form a functional spindle had

remained elusive. Moreover, the exact role of Golgi-based microtubule nucleation in shaping the

mitotic microtubule network had not been characterized.

Here, we describe a pathway responsible for driving astral microtubule formation and spindle

orientation. We show that TPX2 promotes astral microtubule growth around the spindle poles

through Aurora A kinase. TPX2 becomes locally activated by GM130, which sequesters

importin α from TPX2 on mitotic Golgi membranes around the spindle poles. This sequestration-

activation process is triggered by Cdk1-mediated phosphorylation of importin α at Ser62. Mitotic

phosphorylation of importin α weakens its interaction with TPX2 but enhances its binding to

GM130, thereby enabling competition-based activation of TPX2. This Golgi-based TPX2

activation is required for astral microtubule growth from the spindle poles, since suppressing

importin α phosphorylation results in diminished astral microtubule filaments and misorientation

of the spindle.

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Results

Importin α is mitotically phosphorylated by Cdk1 at serine 62

During mitosis, importin α silences several essential spindle assembly factors (such as TPX2)

in the cytoplasm by binding to their nuclear localization sequence (NLS). It had been unclear

what mechanism enables importin α to preferentially recognize these spindle assembly factors in

the presence of other abundant NLS-containing proteins that are released upon nuclear

breakdown at mitotic entry. Previous studies showed that post-translational modifications of

importin α, such as phosphorylation by casein kinase 2, can modulate its activity (Hachet et al.,

2004; Brownlee and Heald, 2019). We hypothesized that mitotic phosphorylation enables

importin α to specifically regulate spindle assembly factors during M-phase. To examine if

importin α is mitotically modified, we separated lysates from and mitotic HeLa cells

on a Phos-tag acrylamide gel, on which phosphorylated proteins migrate more slowly (Figure

1A). By immunoblotting, we observed that the band corresponding to importin α from mitotic

lysate was shifted upward relative to that of interphase lysate, as was the positive control

GRASP55 (Xiang and Wang, 2010). To further establish if the decreased mobility of mitotic

importin α was induced by phosphorylation, we incubated mitotic lysate with λ phosphatase

before analyzing the gel-shift response. Phosphatase treatment abolished the band shift of both

GRASP55 and importin α, demonstrating that importin α is indeed mitotically phosphorylated.

Next, we applied mass spectrometry to identify mitosis-specific phosphorylation sites of

importin α. To this end, we generated a HeLa cell line that stably expresses FLAG-tagged

importin α in a doxycycline-inducible manner, and then isolated tagged importin α from the

interphase and mitotic cell lysates. Mass spectrometry analysis revealed a unique phosphorylated

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site for mitotic importin α, which is located on a fragment comprised of residues 52-68 (Figure

1B).

Importin α binds its substrates and forms a ternary complex with importin ß. In the absence

of NLS-containing substrates, the autoinhibitory domain (IBB) of importin α folds back into its

NLS-binding groove (Figure 1B). Importin ß can bind to the IBB domain of importin α to open

up its NLS-binding region. The phosphorylated fragment we identified (residues 52-68) is

situated between the IBB domain and the substrate-binding domain. This importin α peptide

(NVSSFPDDATSPLQENR) contains several Ser and Thr residues as potential phosphorylation

sites, but harbors only one Ser/Thr-Pro consensus motif for the master mitotic kinase Cdk1-

cyclinB1 (at Ser62). Since this Ser-Pro motif is highly conserved (Figure 1C), we reasoned that

Ser62 is mitotically phosphorylated by Cdk1. To validate this supposition, we generated stable

HeLa cell lines that inducibly express FLAG-tagged wild type importin α (WT) or the importin α

S62A mutant protein, and then analyzed their interphase and mitotic lysates on a Phos-tag gel

(Figure 1D). We found that the mobility of WT importin α from mitotic lysate was reduced

compared to that of interphase lysate, whereas the S62A mutant from mitotic lysate migrated at

the same speed as from interphase lysate. Next, we determined if importin α is mitotically

phosphorylated by Cdk1. We incubated recombinant importin α with mitotic extract in the

presence or absence of the Cdk1 inhibitor Purvalanol A. Phosphorylation was then detected by

blotting with biotinylated Phos-tag combined with streptavidin-conjugated HRP (Figure 1E).

Phos-tag biotin specifically labeled the phosphorylated importin α in mitotic extracts, which was

abolished upon treatment of extracts with the Cdk1 inhibitor Purvalanol A. Taken together, these

results show that importin α is phosphorylated at serine 62 during mitosis by Cdk1.

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Impaired importin α phosphorylation causes mitotic defects

We then sought to determine the effects of importin α phosphorylation on mitotic progression.

To rule out potential chronic effects of expressing phospho-deficient mutant importin α, we

generated stable normal rat kidney (NRK) cell lines that express human FLAG-tagged wild type

importin α (hereafter referred to as “WT-expressing”) or mutant importin α S62A (hereafter

referred to as “S62A-expressing”) upon doxycycline treatment. These importin α-expressing

cells were synchronized and released from a thymidine G1/S block, and then their progression

into M-phase was followed by phase contrast time-lapse microcopy. Average time to mitotic

entry of WT- and S62A-expressing cells was comparable, indicating that ectopic expression of

importin α does not impact interphase progression or the G2/M transition (Figure 2A). However,

the time from cell rounding to onset (as a measure for the duration of mitosis) was

prolonged by 13% (or 5 min) in S62A-expressing cells compared to WT-expressing cells (Figure

2B). Moreover, whereas WT-expressing cells divided normally, 21.2% of mutant cells with

phospho-deficient importin α S62A exhibited cytokinesis defects, often generating more than

two daughter cells (Figures 2C and 2D).

Mitotic phosphorylation of importin α controls spindle orientation

To further investigate the effects of mutant importin α S62A on mitosis, we analyzed the 3D-

orientation of metaphase spindle using confocal microscopy. Cells arrested in metaphase were

double-stained for α- and γ-tubulin to label spindle microtubules and mark the spindle

poles, respectively. Then we measured spindle length and calculated spindle orientation along

the z-axis (Figure 3A). We found that average distances between the two spindle poles did not

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differ significantly between WT- and S62A-expressing cells (Figures 3B and 3D), indicating that

the overall structure of the spindle and the dynamics of spindle microtubule fibers were not

affected (Baudoin and Cimini, 2018). Notably, in WT-expressing cells, both spindle poles were

detected within the same z-section and were aligned parallel to the substratum. In contrast, the

spindle poles of S62A-expressing cells were located in different z-planes, revealing that the

spindle axis was tilted relative to the coverslip surface. The angle between the coverslip surface

and the plane in which the two spindle poles resided showed an aberrant 13° tilt of the spindle

axis in S62A-expressing cells (Figure 3C). These results indicate that expression of phospho-

deficient importin α S62A alters the orientation of the mitotic spindle and affects cell division.

Mutant importin α S62A inhibits astral microtubule growth

Proper orientation of the spindle depends on the astral microtubules that connect the spindle

to the cell cortex (Lu and Johnston, 2013). To decipher if astral microtubules in cells expressing

mutant importin α S62A were compromised, we arrested the cells in metaphase using MG132

and then investigated the microtubule networks by confocal microscopy. To better visualize the

astral microtubules, which are less abundant and dimmer compared to interpolar and kinetochore

microtubules, we increased image exposure. We found that the astral microtubule network

originating at steady-state spindle poles of S62A-expressing cells was less dense and the

filaments were shorter relative to WT-expressing cells (Figure 4A). Furthermore, the relative

fluorescence intensity of α-tubulin staining in the astral area of S62A-expressing cells was

diminished by 30% compared to WT cells (reduced from 9% to 6%, Figure 4B).

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To determine if the S62A mutation affects initiation of astral microtubules, we performed a

microtubule regrowth assay upon cold-induced depolymerization (Figure 4C). Cells arrested in

metaphase were placed on ice for 5 min, resulting in full depolymerization of astral microtubules

(Figure 4D). The cells were then shifted to 37 ºC for 10 min to allow microtubule regrowth and

stained for α-tubulin. This assay showed that the astral microtubule network in WT-expressing

cells was restored to steady-state conditions upon temperature shift, whereas astral microtubule

regrowth was reduced by 25% in S62A-expressing cells (Figure 4E).

To further test if the shorter and less abundant astral microtubule network in S62A-

expressing cells was due to diminished microtubule growth, we performed another microtubule

regrowth assay and stained for EB1 (Figure 4F). EB1 preferentially localizes at the tips of

growing microtubules and functions as a marker for growing microtubule filaments. Consistent

with the tubulin staining (Figure 4E), EB1 signal intensity on astral filaments in S62A-

expressing cells was reduced by 29% (from 16.4% to 11.7% of the EB1 signal in the astral area)

(Figure 4G). Furthermore, the EB1 comets did not emanate as far from the spindle poles of

S62A-expressing cells (2.7 µm) as it did in WT-expressing cells (4.2 μm) (Figure 4H). These

results demonstrate that expression of mutant importin α S62A impairs astral microtubule growth

and dynamics, resulting in spindle misorientation.

TPX2 is required for astral microtubule formation

During mitosis, importin α silences the activity of the spindle assembly factor TPX2 in the

cytoplasm. Upon release from importin α inhibition, TPX2 binds to and activates Aurora A, an

essential kinase for spindle assembly (Garrido and Vernos, 2016). Previously, we demonstrated

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that mitotic Golgi membranes can initiate localized microtubule assembly by liberating TPX2

from importin α inhibition (Wei et al., 2015). Given that mitotic Golgi membranes accumulate

around the spindle poles (Wei and Seemann, 2009a), we wondered if TPX2 binding to Aurora A

drives astral microtubule formation. To test this possibility, we took advantage of AurkinA, a

recently developed small-molecule inhibitor that specifically blocks the interaction of TPX2 with

Aurora A (Figure 5A) (Janeček et al., 2016). We first tested the overall efficacy of the inhibitor

on the spindle microtubules. Cells arrested in metaphase were treated with 200 µM AurkinA for

0, 15 or 30 min, and then immuno-stained for tubulin (Figure 5B). We found that prolonged

AurkinA treatment (for 30 min) weakened the microtubule filament network, whereas 15-min

AurkinA incubation mainly impacted astral microtubules compared to spindle filaments (Figures

5C and 5D). Furthermore, the 15 min AurkinA treatment reduced the lengths of astral

microtubules, as well as EB1 signal intensity and density in the astral microtubule area (Figures

5E and 5F). These results indicate that TPX2 binding to Aurora A kinase is required for astral

microtubule formation.

GM130 regulates astral microtubule growth via phosphorylated importin α

Upon entry into M-phase, the Golgi ribbon is swiftly disassembled into a collection of

vesicles and clusters of membranes (termed mitotic Golgi clusters). By metaphase, these mitotic

Golgi clusters are concentrated around the two spindle poles in the vicinity of astral microtubule

filaments (Jokitalo et al., 2001). Competition for importin α binding by GM130 on Golgi

membranes activates the microtubule initiation role of TPX2 (Wei et al., 2015). Therefore, we

tested if the mitotic Golgi clusters can initiate astral microtubule polymerization through TPX2

and if, in turn, the astral microtubules are important for proper localization of mitotic Golgi

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clusters. We found that expression of WT or S62A-mutant importin α in interphase NRK cells

did not affect the morphology or localization of the Golgi apparatus in the perinuclear region of

those cells (Figure 6A). However, during mitosis, we observed that expression of importin α

S62A not only diminished astral microtubules (as shown in Figure 4), but mitotic Golgi clusters

were also displaced from the spindle pole area and were reduced in size in S62A-expressing cells

(Figure 6B).

We then sought to acutely interfere with the microtubule initiation capacity of the mitotic

Golgi clusters. The microtubule initiation activity of GM130 can be blocked by microinjection of

inhibitory anti-GM130 antibodies (Wei et al., 2015). We microinjected affinity-purified GM130

antibodies or control IgG into MG132-arrested metaphase NRK cells. Then, 30 min after the

injection, we depolymerized astral microtubules by cold treatment, before shifting the cells to 37

ºC for 10 min to allow filament regrowth (Figure 6C). The cells were then stained for the

injected antibodies (to discriminate the cells), as well as for tubulin and EB1. Blocking GM130

activity phenocopied the effects of AurkinA treatment (Figure 5) by attenuating astral

microtubules (Figures 6C and 6D) and their growth dynamics (as revealed by reduced EB1

labeling; Figures 6E and 6F). Consistent with expression of mutant importin α S62A, we further

noted from the staining patterns of injected GM130 antibodies that Golgi clusters were more

fragmented and delocalized from the spindle pole area (Figures 6C and 6E). These results

suggest that the GM130-TPX2 pathway activates astral microtubule assembly and is enabled by

importin α phosphorylation.

Importin α is recruited to Golgi membranes during mitosis via GM130 (Wei et al., 2015). If

mitotic phosphorylation of importin α is required for binding to GM130, then importin α S62A

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should not associate with mitotic Golgi membranes. We tested this possibility using velocity

gradient centrifugation to separate smaller mitotic Golgi vesicles from larger membranes (Jesch

et al., 2001; Seemann et al., 2002). HeLa cells arrested in mitosis by means of the Eg5 kinesin

inhibitor S-Trityl-L-Cysteine were collected by shake-off and mechanically lysed using a ball

bearing homogenizer. The post-chromosomal supernatant was then centrifuged through a linear

5%-25% glycerol gradient and then the membranes of collected fractions were immunoblotted

for GM130 and FLAG-tagged importin α (Figure 6G). Consistent with our previous report (Wei

et al., 2015), WT importin α co-migrated with GM130 in the top fractions of the gradient,

demonstrating that importin α had been recruited to mitotic Golgi membranes. In contrast,

phospho-deficient importin α S62A did not associate with mitotic Golgi membranes in the top

fractions of the gradient. We also observed that the GM130-containing membrane fractions were

shifted upwards to the lighter membranes in the gradient, in line with our immunofluorescence

data showing that smaller mitotic Golgi clusters formed in S62A-expressing cells (Figure 6B).

Some of the importin α remained in the bottom fraction of the gradient comprising the bulk of

the cellular membranes, consistent with previous reports that importin α also associates with the

nuclear envelope and the plasma membrane during mitosis (Hachet et al., 2004; Brownlee and

Heald, 2019).

To further assess the phosphorylation dependency of the importin α-substrate interaction, we

pulled down FLAG-tagged WT or importin α S62A from mitotic lysates and probed for

association of endogenous GM130 and TPX2 by means of immunoblotting (Figure 6H). The

results showed that TPX2 binds much more strongly to importin α S62A than to phosphorylated

WT importin α. Conversely, binding of GM130 to phosphorylated WT importin α was greatly

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enhanced relative to phospho-deficient importin α S62A. These results evidence that

phosphorylation of importin α shifts its substrate preference from TPX2 to GM130, allowing

GM130 on mitotic Golgi membranes to sequester importin α from TPX2 and activate Aurora A

to regulate astral microtubules and spindle orientation.

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Discussion

Here, we report that TPX2 activation regulates astral microtubule dynamics and is essential

for correct spindle orientation (Figure 7). Upon mitotic entry, phosphorylation of importin α at

Ser62 by Cdk1 induces a switch in substrate preference from TPX2 to GM130 and thereby

enables GM130 to activate TPX2 through direct competition. The phospho-incompetent importin

α S62A point mutant prevents GM130 from activating TPX2, reducing astral microtubules and

causing spindle misorientation. Furthermore, the characteristic clustering of mitotic Golgi

membranes at the spindle poles is astral microtubule-dependent and requires TPX2 activation.

Spindle orientation is important in determining cell fate and function. For example, in

mammalian skin epidermal cells, the orientation of the spindle defines the division plane and,

consequently, cell fate (Barr and Gruneberg, 2017). Embryonic epidermal progenitor cells

initially proliferate in a single layer by dividing parallel to the basement membrane. During

stratification, the cells may also divide perpendicular to the basement membrane, generating one

daughter cell that remains proliferative in the basal layer and another that is destined for terminal

differentiation. Therefore, spindle orientation must be tightly controlled, as defects in spindle

orientation trigger unbalanced growth and differentiation, outcomes linked to hyperproliferation

and cancer (Ragkousi and Gibson, 2014; Asare et al., 2017). Similarly, aberrant spindle

orientation due to impairment of astral microtubule-cortex connectivity in Drosophila alters the

balance between neural progenitor expansion versus differentiation, leading to defects in

neuroblast homeostasis (Cabernard and Doe, 2009). Moreover, knockdown of ASPM (abnormal

spindle-like microcephaly-associated ), which directly contributes to the growth of astral

microtubules, causes abnormal spindle orientation in neuroepithelial progenitor cells, and

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mutation of ASPM is the most common defect observed in human autosomal recessive primary

microcephaly (Fish et al., 2006; Gai et al., 2016). Interestingly, similar defects have been

reported for GM130 mutations in vivo. Knockdown of GM130 in Zebrafish leads to

microcephaly (Shamseldin et al., 2016). The Purkinje cells of GM130-deficient mice exhibit

neurodegeneration (Liu et al., 2016), and a patient with a homozygous loss-of-function mutation

of GM130 developed neuromuscular disorder including progressive microcephaly (Shamseldin

et al., 2016). These phenotypes have been broadly assigned to defective secretory pathways in

which GM130 functions as a vesicle tethering factor during interphase. However, our present

work shows that GM130 regulates spindle orientation, suggesting that deficiencies in GM130

mitotic function might further contribute to these neuronal disorders through impaired cell

division and development.

Ran-dependent TPX2 activation takes place at the chromosomes. Analysis of RanGTP

distribution in mitotic cells detected a low concentration of RanGTP in the astral area where

Ran-mediated activation of TPX2 is unlikely to happen (Kaláb et al., 2006). Therefore, initiation

and growth of astral microtubules has often been attributed solely to the centrosomes and

microtubule-stabilizing proteins. Our findings have identified TPX2 as a key molecule in

promoting astral microtubule growth. The direct competition-based activation of TPX2 on Golgi

clusters does not require RanGTP and facilitates microtubule organization in a distinct

spatiotemporal fashion.

Our results also indicate that phosphorylation-deficient importin α S62A inhibits TPX2 from

being activated by GM130 at the spindle poles, leading to diminished astral microtubule density

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that, in turn, misorients the spindle. Since importin α modulates several mitotic regulators, other

mitotic events could also be affected by importin α S62A, such as the cytokinesis defects we

report here. However, our observed phenotype of spindle misorientation induced by importin α

S62A should mainly arise from compromised astral microtubule dynamics. We did not record

spindle oscillation, which has often been reported for cells with spindle-cell cortex junction

defects (Kotak and Gönczy, 2014; Petry, 2016). The centrosomal protein ASPM targets the

citron kinase CITK to the spindle poles, where it specifically regulates astral microtubule

dynamics (Gai et al., 2016). In ASPM-knockdown cells, spindle length is unchanged and spindle

oscillation has not been observed. Moreover, the metaphase to anaphase transition is delayed by

five minutes in such cells and the spindle is misoriented (Gai et al., 2016). Expression of

importin α S62A essentially phenocopied knockdown of ASPM. Therefore, it is reasonable to

conclude that importin α regulates astral microtubule growth by promoting astral microtubule

dynamics from the Golgi clusters at the pole area. Moreover, our acute inhibition of the GM130-

TPX2 pathway slowed down microtubule growth, prompting us to conclude that the GM130-

TPX2 pathway is an important element of the mechanism controlling astral microtubule

dynamics.

Apart from the Golgi, other membrane components have been reported to influence astral

microtubule dynamics (Barr and Gruneberg, 2017). Recycling endosomes can promote

microtubule growth in mitotic extracts, and knockdown of the recycling endosomal protein

Rab11 results in compromised astral microtubules and rotating spindles (Hehnly and Doxsey,

2014). Based on those findings, it has been proposed that the poleward movement of recycling

endosomes transports microtubule-nucleating material like γ-tubulin and GCP4 protein to the

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centrosomes and facilitates the polymerization of astral microtubules. Peroxisomes also play a

role in orienting the spindle. Knockdown of peroxisomal protein Pex11b results in diminished

cell cortex localization of NuMA, contributing to spindle oscillation and misorientation (Asare et

al., 2017). Here, we report mitotic Golgi clusters as being membrane organelles that regulate

spindle orientation. We showed in a previous study that the association between mitotic Golgi

membranes and the spindle is required for a single Golgi ribbon to reform upon mitotic exit (Wei

and Seemann, 2009a). In the current study, we have demonstrated that the association of mitotic

Golgi clusters with astral microtubules originates from the ability of the former to nucleate

microtubules. When we blocked GM130-triggered TPX2 activation by microinjecting inhibitory

antibodies, the Golgi clusters became delocalized from the spindle poles and dispersed. In

addition, expression of importin α S62A diminishes the size of these Golgi clusters. Although it

is still not clear how mitotic Golgi clusters are organized in vivo, our results suggest that the

microtubule network plays an integral role in maintaining the morphology and localization of the

mitotic Golgi clusters at the spindle poles.

Post-translational modifications of importin family proteins affect their binding affinity to

their substrates, influencing the speed of cargo transport into the nucleus during interphase.

Importin α is repurposed as a spindle modulator during mitosis, but it was not clear how it

specifically recognizes spindle assembly factors at this time given the amount of NLS-containing

proteins that are released into the cytoplasm upon nuclear envelope breakdown (Giesecke and

Stewart, 2010). Our study shows that Cdk1-mediated and mitotic-specific phosphorylation of

importin α at Ser62 shifts its binding preference from TPX2 to GM130. Since importin α

regulates other proteins that play important roles in mitosis, phosphorylation of importin α

18 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

potentially also modulates their activities. While we were preparing this manuscript, it was

reported that Ser62 phosphorylation is correlated with decreased binding to TPX2, but the

mechanistic significance at the cellular level was not explored (Guo et al., 2019). A recent report

based on the structure of non-phosphorylated importin α complexed with the N-terminal

fragment of GM130 suggests that, unlike other NLS sequences, GM130 competes with TPX2 for

the minor binding site of the NLS-binding pockets in importin α, thereby preventing other NLS-

containing cargos from activating TPX2 by competitive inhibition (Chang et al., 2019). A more

thorough study on phosphorylation-induced conformational change should help further establish

the exact mechanism by which importin α is regulated and repurposed throughout the .

19 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Materials and methods

Plasmids and cell lines

Human importin α1 (KPNA2) WT or the S62A point mutant was cloned into pET30a for His-

importin α recombinant protein purification. The S62A mutation was generated by quick-change

PCR mutagenesis. Importin α with an N-terminal FLAG tag was cloned into pLVX-TetOne-Puro

(Clontech) to generate stable cell lines. pLVX-FLAG-KPNA2 WT or S62A was co-transfected

with psPAX and pVSVG into HEK293T cells to produce lenti-virus, which was then used to

transduce NRK and HeLa cells. Two days later, we added 5 μg/ml puromycin (RPI) and stable

clones were selected for seven days. Individual colonies were picked and single clones were

isolated by limited dilution. Expression of FLAG-importin α WT or S62A was induced for 16 h

by addition of 1 µg/ml doxycycline (Sigma).

Cell culture and drug treatments

HEK293T, HeLa and normal rat kidney (NRK) cells were cultured at 37 °C and 5% CO2 in

complete growth medium [Dulbecco’s modified Eagle’s medium (Mediatech) supplemented

with 10% cosmic calf serum (HyClone), 100 units/ml penicillin and 100 µg/ml streptomycin].

NRK cells were synchronized at the G1/S phase transition by 16 h treatment with 2 mM

thymidine (Chem-Impex). Then, we washed out the thymidine and added 24 µM 2’-

deoxycytidine (Chem-Impex). Five hours after release from thymidine, we added 10 µM MG132

(Boston Biotech) for 1 h to arrest cells in metaphase.

HeLa cells and HeLa cells expressing FLAG-tagged WT or S62A importin α were synchronized

in mitosis by treatment with 20 µM S-Trityl-L-Cysteine (Acros) for 16 h. The mitotic cells were

20 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

then collected by shake-off, followed by centrifugation. We employed 200 µM AurkinA

(Aobious) in complete culture medium to block the association of TPX2 with Aurora A kinase.

Antibodies

For Western blotting, we used mouse monoclonal antibodies against importin α (BD

Transduction Lab, Cat. #610485, 25 ng/ml) and GAPDH (GA1R, Invitrogen, Cat. #MA5-15738,

20 ng/ml), as well as rabbit polyclonal antibodies against Phospho-Histone H3 (Ser10)

(Millipore, Cat. #06-570, 25 ng/ml), TPX2 (Proteintech, Cat. #11741-1-AP, 150 ng/ml), FLAG-

tag (Sigma, Cat. #F7425, 100 ng/ml), and GRASP55 (Rich, serum 1:5000) (Wei and Seemann,

2009a).

For immunofluorescence, we used mouse monoclonal antibodies against EB1 (BD Transduction

Lab, Cat. #61534, 1 μg/ml), α-tubulin (TAT1; supernatant 1:200) (Woods et al., 1989), and

GM130 (NN2C10, 5 µg/ml, used in Figures 6A and 6B) (Seemann et al., 2000), rabbit

polyclonal antibody against g-tubulin (Sigma, Cat.# T3559), as well as rat monoclonal antibody

against α-tubulin (Chemicon, Cat. #MAB1860, 5 µg/ml). For microinjection, we used affinity-

purified rabbit anti-GM130 (Wei et al., 2015) and rabbit IgG (Sigma).

Secondary antibodies were as follows: Alexa Fluor 488- or Alexa Fluor 594-conjugated to goat

anti-mouse or goat anti-rabbit IgG (Invitrogen), Alexa Fluor 568-conjugated to goat anti-rat IgG

(Invitrogen), HRP-conjugated goat anti-rabbit (Jackson ImmunoResearch), HRP-conjugated goat

anti-mouse IgG (Invitrogen).

21 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Microinjection

NRK cells grown on glass coverslips were microinjected using a FemtoJet system (Eppendorf)

and a micromanipulator 5171 (Eppendorf) attached to an Eclipse TE300 inverted microscope

(Nikon). The cells were switched to complete growth medium supplemented with 50 mM

HEPES/KOH pH 7.4 before injections. The cells were microinjected with 2 mg/ml affinity-

purified rabbit anti-GM130 or 2 mg/ml control IgG in H/K buffer (20 mM HEPES/KOH pH 7.4,

50 mM KOAc). Injected cells were incubated at 37 °C for 30 min and then subjected to an astral

regrowth assay. Cells were then fixed in -20 °C methanol for EB1 staining or in microtubule-

stabilizing fixative to stain for α-tubulin.

Immunofluorescence

For the astral microtubule regrowth assay, we replaced the medium with cold complete growth

medium for 5 min to depolymerize astral microtubules and then changed it back to 37 °C

complete growth medium and continued incubating the cells at 37 °C for 10 min to allow

microtubule regrowth. To stain for aster microtubules, the cells were fixed for 20 min at 37 °C in

microtubule-stabilizing fixative (3.7% formaldehyde, 0.1% glutaraldehyde, 60 mM PIPES, 25

mM HEPES, 2 mM MgCl2 and 10 mM EGTA, pH 6.9). The reaction was quenched for 5 min

with sodium borohydride in 20 mM Tris pH 7.4 and 150 mM NaCl, before being permeabilized,

washed three times with TBS-Tx (20 mM Tris pH 7.4, 150 mM NaCl and 0.1% Triton X-100),

and blocked with 1 mg/ml BSA in TBS-Tx at room temperature for 30 min.

For all other immunofluorescence stainings, the cells were fixed and permeabilized for 15 min in

methanol at -20 °C, then incubated with respective antibodies at 37 °C for 30 min, followed by

Alexa-Fluor-conjugated secondary antibodies. DNA was stained for 10 min at room temperature

22 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

with 1 µg/ml Hoechst 33342 (Invitrogen) in PBS and cells were embedded in Mowiol

(Calbiochem) mounting solution (Wei and Seemann, 2009b).

Subcellular fractionation

Mitotic HeLa cells expressing WT or importin α S62A were mechanically lysed using a ball

bearing homogenizer with a clearance of 12 µm (Isobiotec). The post-chromosomal supernatant

(PCS) was collected by centrifugation for 5 min at 1000 g at 4 °C, loaded onto a continuous 5%-

25% glycerol gradient, and centrifuged for 30 min in a SW55Ti rotor (Beckman) at 35,000 rpm

at 4 °C. Ten 0.5 ml fractions were collected from the top of the gradient, diluted with 1 ml of 20

mM HEPES/KOH pH 7.4, and membranes were collected by centrifugation for 30 min at

100,000 g at 4 °C. The supernatant was discarded, and the membrane pellets were resuspended in

SDS sample buffer for further analysis by Western blotting.

Co-immunoprecipitation

Interphase or mitotic HeLa cells expressing FLAG-importin α WT or S62A mutants were lysed

for 20 min at 4 °C in lysis buffer [10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1%

TritonX-100, cOmplete protease inhibitor cocktail (Roche), 1 mM DTT, 10 mM β-

glycerophosphate, 1 mM Na3VO4 and 10 mM NaF]. Lysates were cleared by centrifugation at

16,000 g for 10 min at 4 °C and then incubated with 10 µl anti-FLAG M2-Sepharose beads

(Sigma) for 60 min at 4 °C. Beads were then washed three times with lysis buffer and boiled in

SDS sample buffer to elute proteins for Western blotting analysis. For mass spectrometry,

FLAG-importin α purified from mitotic and interphase HeLa cells was eluted from the beads

23 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

using 300 µg/ml 3xFLAG-tag peptide (Apexbio). The 58 kDa bands were cut from the gel and

processed by the Proteomics Core Facility at UT Southwestern.

Protein purification

BL21(DE3) competent E. coli were transformed with pET30a-importin α (WT, S62A). Single

clones were inoculated into 20 ml LB medium and grown overnight at 37 °C. The 20 ml cultures

were then inoculated into 1000 ml LB to allow the bacteria to grow to OD600=0.6-0.8. Protein

expression was induced by addition of 0.2 mM IPTG and incubation for 18 h at 16 °C. Bacteria

were collected by centrifugation, resuspended in high salt buffer (HSB) [PBS supplemented with

1 M NaCl, cOmplete protease inhibitor cocktail (Roche), 1 mM DTT, 1 mM PMSF] and lysed

using a French Press. Nickel-NTA agarose beads (Qiagen) were used to purify His-importin α (1

ml slurry for 1000 ml culture). The beads were then washed with HSB and directly used for in

vitro phosphorylation (5 μl slurry per experiment).

In-vitro phosphorylation assay

Mitotic HeLa cells were lysed for 20 min at 4 °C in lysis buffer [10 mM Tris pH 7.4, 150 mM

NaCl, 1 mM EDTA, 1% TritonX-100, cOmplete protease inhibitor cocktail (Roche), 1 mM DTT,

10 mM β-glycerophosphate, 1 mM Na3VO4 and 10 mM NaF], and then the lysates were cleared

by centrifugation at 16,000 g for 10 min at 4 °C. Nickel beads coated with recombinant His-

tagged importin α were incubated at 30 °C for 30 min with pre-cleared mitotic lysate

supplemented with 2 mM ATP pH 7.2. The Cdk1 kinase inhibitor Purvalanol A (LC Labs) was

used at a concentration of 0.5 mM. Beads were then washed three times with HBS, eluted by

boiling in SDS sample buffer, and analyzed by Phos-tag biotin blotting.

24 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Phos-tag gel and Phos-tag biotin

To detect importin α phosphorylation via in vitro phosphorylation assay, protein samples were

separated by SDS-PAGE on a 10% gel and transferred to PVDF membrane. The PVDF

membrane was then washed with TBST for 20 min before incubation with Phos-tag biotin

solution [TBST supplemented with 6 µM ZnCl2, 2 µM Phos-tag Biotin (ApexBio) and 2 ng/ml

streptavidin-HRP (Jackson ImmunoResearch)]. After 30 min incubation, the blot was developed

by ECL detection.

To detect importin α phosphorylation on Phos-tag gels, mitotic or interphase cells were lysed for

20 min at 4 °C in lysis buffer (without EDTA) and cleared by centrifugation at 16,000 g for 10

min at 4 °C. For phosphatase treatment, 40 µl of the cleared lysates were incubated with 1 µl

Lambda Protein Phosphatase (l phosphatase) (New England Biolabs), 5 µl 10x buffer for

Protein MetalloPhosphatases (New England Biolabs), and 5 µl 10 mM MnCl2 for 30 min at 30

°C. Proteins were separated on a 10% SDS gel containing 20 μM Phos-tag acrylamide

(ApexBio) and 100 µM MnCl2. The gel was then washed for 1 h in transfer buffer containing 1

mM EDTA to remove the manganese. Proteins were subsequently transferred to PVDF

membrane for immunoblotting.

Microscopy and quantitation

Confocal microscopy was performed using a Zeiss LSM780 inverted microscope in combination

with a Plan-Apochromat 63x/1.4 objective. Z-sections were captured at 0.5 µm intervals and

maximum intensity projections are presented. Spindle angle was calculated using the formula α =

arctan (b/a), where b = 0.5 μm multiplied by the number of z sections between the spindle poles

25 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

and a = the distance between the projections of the two spindle poles on the x-y plane. Spindle

length (l) was calculated as l2=a2+b2.

Relative fluorescence of astral microtubules was measured in ImageJ (as depicted in Figure 4B)

as the fluorescence signal of the astral microtubule area/total microtubule fluorescence signal of

the cell.

Astral microtubule length was determined by averaging the distance between the tip of the EB1

“comet” and the pole for the three longest filaments from each of the two asters.

Phase-contrast time-lapse microscopy was performed using an Axiovert 200M microscope

(Zeiss) with an LD-A-PLAN 20x/0.3 Ph1 objective (Zeiss) and a Retiga 2000R CCD camera

(QImaging) using MetaMorph 7.1.3 software (Molecular Devices). NRK cells expressing WT

importin α or mutant importin α S62A released from thymidine blocking were changed to CO2-

independent medium (Invitrogen), 10% cosmic calf serum (HyClone), 2 mM GultaMax

(Invitrogen) and 100 units/ml penicillin and 100 μg/ml streptomycin. Phase contrast images were

captured at 10 min intervals for 12 h, and cell cycle progression was analyzed using MetaMorph

7.1.3 (Molecular Devices) and ImageJ. The duration from G1/S phase to mitosis was determined

as being from 30 min after thymidine washout until rounding of the cells. The duration of mitosis

was measured as the time from rounding of the cells to onset of cytokinesis.

Image analysis was performed using ImageJ 2.0. Statistical analyses were conducted using Prism

8.3 software (GraphPad). Data shown are from three or more independent experiments. Error

bars represent SEM. Statistical significance was assessed by Student’s t tests.

26 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Acknowledgements

We thank Yijun Zhang for comments on the manuscript and other suggestions, Gianni Guizzunti

for insightful discussions, the Proteomics Core Facility for mass spectrometry analysis and the

Light Microscopy Facility at the University of Texas Southwestern Medical Center for imaging

support. J-H.W. is supported by an Academia Sinica Career Development Award (AS-CDA-109-

L02). This work was supported by grants from the NIH (GM096070) and the Welch Foundation

(I-1910) to J.S.

Author contributions

H.G., J-H.W. and J.S. designed the project and wrote the manuscript. H.G. and J.S. performed

the experiments, analyzed the data, and prepared the figures.

Competing interest

The authors declare no competing interests.

27 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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Figure Legends

Figure 1. Importin α is mitotically phosphorylated at serine 62 by Cdk1.

(A) Importin α is phosphorylated during mitosis. Cell lysates from interphase (int) and mitotic

(mit) HeLa cells, as well as λ phosphatase-treated mitotic lysate, were separated on a 10% SDS

gel containing 25 μM Phos-tag acrylamide and immunoblotted for importin α. GRASP55 and

GADPH were separated by regular SDS-PAGE.

(B) Schematic illustration of the location of the mitotically-phosphorylated peptide identified by

mass spectrometry. The identified peptide (residues 52-68) harbors the mitosis-specific

phosphorylation site of importin α and is located between the importin β-binding domain and the

NLS-binding domain.

(C) Within the identified peptide sequence, residue serine 62 is conserved among higher

eukaryotes. The serine/proline sequence resembles a Cdk1 phosphorylation consensus motif.

(D) Importin α is phosphorylated at serine 62. Interphase or mitotic cell lysates from cells

expressing FLAG-tagged WT importin α or importin α S62A were separated on a Phos-tag

acrylamide gel and immunoblotted for FLAG. Phospho-Histone H3 and GAPDH were separated

by SDS-PAGE.

(E) Importin α phosphorylation is blocked by the Cdk1 inhibitor Purvalanol A. Beads coated

with recombinant full-length His-tagged importin α were incubated with mitotic HeLa extract in

the presence or absence of Purvalanol A. The beads were then washed by high salt buffer (PBS,

1M NaCl), and importin α was eluted by boiling in SDS sample buffer. Phosphorylation was

detected by blotting with Phos-tag biotin and streptavidin-HRP.

32 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2. Importin α S62A induces mitotic defects.

NRK cells non-induced or induced to express WT importin α or importin α S62A were

synchronized at the G1/S transition by means of thymidine treatment for 16 h. The cells were

then released from the thymidine block and cell cycle progression was monitored by time-lapse

phase contrast microscopy.

(A) Importin α expression does not affect mitotic entry. The duration between release from G1/S

arrest to mitotic entry is plotted (n>90). Error bars represent SEM.

(B) Importin α S62A-expressing cells exhibit prolonged mitotic duration (by 5 min; n>90).

(C and D) NRK cells expressing importin α S62A exhibit cytokinesis defects (n>90). Error bars

represent SEM. Scale bar = 20 µm.

Figure 3. Phosphorylation of importin α controls spindle orientation.

(A) Schematic illustration of spindle orientation in tissue culture cells. The axis of a normal

spindle is oriented parallel to the coverslip, whereas the axis of a misoriented spindle is tilted.

The angle α of spindle tilt was calculated as: α=arctan (b/a). Spindle length (l) was calculated as

the 3D distance between the spindle poles (l2=a2+b2).

(B) Importin α S62A causes misorientation of the spindle. NRK cells expressing WT importin α

or importin α S62A were arrested at metaphase using MG132 and then dual-stained for α-tubulin

and γ-tubulin to label spindle microtubules and the spindle poles, respectively. Z-sections were

captured by confocal microscopy. Orthogonal sections through the z stacks along the x-axis

(bottom panels) indicate the relative spindle pole localization on the z-axis. Scale bar = 10 μm.

(C) Importin α S62A results in tilted spindles. The spindle tilt angle α was calculated as shown in

(A). n>30, error bars represent SEM.

33 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

(D) Importin α S62A does not alter spindle length. The spindle length l was determined as shown

in (A). n>30, error bars represent SEM.

Figure 4. Importin α S62A inhibits microtubule growth.

(A) Importin α S62A compromises astral microtubules. NRK cells expressing WT importin α or

importin α S62A were arrested at metaphase using MG132 for 1 h and stained for α-tubulin.

Maximum intensity projections of confocal z-sections are shown. Enlargements of the astral

microtubule area are shown in the panels at right.

(B) Quantitation of (A). Astral microtubule fluorescence was measured as the percentage of

astral microtubule intensity relative to the total microtubule intensity, as shown in the schematic.

n>30, error bars represent SEM.

(C) Schematic illustration of the cold-induced astral microtubule regrowth assay. Astral

microtubules were depolymerized by 5 min of cold treatment, and then allowed to regrow by

shifting the cells back to 37 °C for 10 min.

(D) Importin α S62A inhibits astral microtubule regrowth upon cold treatment. NRK cells

arrested at metaphase using MG132 were placed on ice for 5 min to depolymerize astral

microtubules but not spindle filaments. After 10 min regrowth at 37 °C, cells were fixed and

stained for α tubulin. Confocal microscopy images represent maximum intensity projections.

(E) Astral microtubule fluorescence was calculated as in (B). n>30, error bars represent SEM.

(F) Importin α S62A inhibits EB1 localization to the astral area. EB1 was stained following the

procedure in (C and D). Maximum intensity projections are shown.

(G) EB1 fluorescence was calculated as for astral microtubule intensity. n>30, error bars

represent SEM.

34 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

(H) Importin α S62A shortens astral microtubule length. The lengths of astral microtubules were

determined as the distance between the tip of an EB1 signal comet and the spindle pole. For each

cell, the average length of the three longest filaments from each pole was used to represent

nascent microtubule length. n>30, error bars represent SEM. All scale bars = 5 µm.

Figure 5. TPX2 is required for astral microtubule formation.

(A) AurkinA is a small molecule inhibitor that specifically blocks the interaction of TPX2 with

Aurora A kinase.

(B) AurkinA alters spindle organization. NRK cells arrested at metaphase using MG132 for 1 h

were treated with AurkinA for 0, 15 or 30 min and then fixed and stained for α-tubulin.

Maximum intensity projections are shown.

(C) AurkinA compromises astral microtubules. After 15 min of AurkinA treatment, NRK cells

were fixed and stained for α-tubulin.

(D) Quantitation of (C). n>30, error bars represent SEM.

(E) AurkinA inhibits astral microtubule growth. After 15 min of Aurkin A treatment, NRK cells

were fixed and stained for EB1.

(F) Quantitation of (E). n>30, error bars represent SEM.

All scale bars = 5 µm. Maximum intensity projections of confocal z-sections are shown.

35 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 6. GM130 regulates astral microtubule growth via phosphorylated importin α.

(A) Importin α S62A does not alter Golgi structure or the microtubule network during interphase.

Interphase NRK cells expressing WT importin α or importin α S62A were fixed and stained for

α-tubulin and the Golgi membrane protein GM130. Maximum intensity projections are shown.

Scale bar = 10 µm.

(B) Importin α S62A delocalizes mitotic Golgi clusters from the spindle pole area. NRK cells

expressing WT importin α or importin α S62A were arrested at metaphase using MG132, and

then fixed and stained for GM130 and α-tubulin. Maximum intensity projections are shown.

Scale bar =10 µm.

(C and E) Blocking the GM130-importin α interaction inhibits astral microtubule growth and

delocalizes Golgi clusters from the spindle poles. NRK cells were arrested at metaphase for 1 h

and microinjected with inhibitory antibody against GM130 or with control IgG. Injected cells

were incubated for another 30 min, followed by cold treatment for 5 min. They were then

allowed to regrow microtubules for 10 min, before fixation and staining for α-tubulin, EB1 and

injected IgG. Maximum intensity projections are shown. Scale bars = 5 µm.

(D and F) Quantitation of astral microtubule fluorescence and astral EB1 fluorescence from (C

and E). n>30, error bars represent SEM.

(G) Importin α S62A does not associate with mitotic Golgi membranes. Post-chromosomal

supernatant of mitotic HeLa cells expressing WT importin α or importin α S62A was centrifuged

through a linear 5%-25% glycerol gradient to separate membrane components by size. Ten

fractions were collected from the top. The membranes from each fraction were collected by

centrifugation and then analyzed by Western blotting.

36 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

(H) Phosphorylation of importin α shifts its binding preference from TPX2 to GM130.

Interphase or mitotic HeLa cells expressing FLAG-tagged WT importin α or importin α S62A

were lysed and incubated with anti-FLAG antibody-conjugated beads. Beads were then washed

with lysis buffer and analyzed by Western blotting.

Figure 7. Model of the TPX2-mediated astral microtubule assembly pathway.

Phosphorylation of importin α at Ser62 by Cdk1 switches its substrate preference from TPX2 to

GM130. This switch enables GM130 on the mitotic Golgi clusters at the spindle poles to locally

sequester importin α from TPX2 through direct competition. TPX2 is thereby relieved from

inhibition, allowing it to bind to and activate Aurora A kinase to drive astral microtubule

assembly and facilitate spindle orientation.

37 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.16.994426; this version posted March 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.